A large fraction of cellular transcription is devoted to the synthesis of ribosomal RNA (rRNA). Our laboratory has developed 2 parallel Drosophila systems in which to study ribosomal RNA (rRNA) metabolism in both dividing and non-dividing cells. The signalling events that upregulate rRNA production in both cell types involved increased calcium and activation of protein kinase C. Antibiotics which alter the levels of nontranslating versus translating ribosomes also effect changes in rRNA levels. Nuclear run-on experiments indicate that, in each case, this regulation appears to be at the level of transcription. Therefore, we propose a series of experiments to investigate the DNA sequences and protein factors that mediate the responses of Drosophila cells to these rRNA regulatory reagents. We have constructed a reporter gene system to investigate the essential regulatory nature of particular rDNA sequences. It reproducibly gives stable transcripts in transient transfection assays. The levels of these transcripts are correctly regulated by calcium phorbol esters and serum. We will construct deletions and rearrangements of the rDNA sequences in the reporter gene system. These DNAs will be returned to cultured cells by transfection and to the Drosophila genome by P-element transformation and will be assayed for their ability to support RNA polymerase I (Pol I) transcription of the reporter. Nuclear extracts from stimulated cells can direct increased, correct transcription of a Pol I template. The extracts also show differential DNA-binding activities on potential regulatory sequences. These experiments will now serve as the basis for an assay system of separated protein fractions as well as allow us to conduct in vitro template commitment assays. Lastly, we will investigate the effects of insertion sequences which inhibit transcription in the rRNA genes in which they are found by placing these sequences in other genomic positions and constructions. In total, these experiments will help us distinguish the sequences and proteins that mediate the regulation of Drosophila rRNA synthesis and determine whether such varied reagents as calcium, serum, phorbol esters, calcium ionophores and protein synthesis reagents control rRNA synthesis by the same or different pathways and whether these pathways are the same in mitotic and nonmitotic cells.